1. Field of the Invention
The present invention relates generally to the fields of chemical cross-linkers, peptide chemistry and DNA carriers. More particularly, the invention provides surprisingly effective cross-linking peptides and peptide-DNA compositions with increased stability and reduced toxicity. Methods of using the peptide-DNA condensates in gene delivery and gene expression are also provided, optionally in combination with matrices, carriers and/or targeting agents.
2. Description of Related Art
Gene therapy is a growing field with far-reaching medical implications. Gene therapy can be used to replace specific genes, as in the correction of a heritable defect, and/or to deliver functionally active therapeutic genes into target cells. Other clinically applicable aspects of nucleic acid delivery involve the application of inhibitory nucleic acids, such as antisense constructs and/or ribozymes, to inhibit aberrant gene products, as in the treatment of cancer.
Initial efforts towards somatic gene therapy relied on indirect means of introducing genes into tissues, i.e., ex vivo gene therapy. In such embodiments, cells are removed from the body, transfected or infected with vectors carrying recombinant genes in vitro, and re-implanted into the body (xe2x80x9cautologous cell transferxe2x80x9d). As an alternative, viral-mediated gene delivery is efficient, but is associated with drawbacks that limit its clinical application.
A variety of nonviral gene delivery carriers have been developed and tested as in vitro transfection agents, used to transiently express foreign DNA in cells in culture. Cationic lipids (Zhang et al., 1997), polylysine peptides (Wagner et al., 1990; Wyman et al., 1997; Morris et al., 1997) and cationic polymers such as polyethylenamine (Ogris et al., 1998; Boussif et al., 1995), bind electrostatically to the phosphate backbone of DNA to form complexes that mediate cellular uptake in culture.
Nonviral gene delivery using various carrier molecules has also been proposed for in vivo use (Wu and Wu, 1988; Wu et al., 1989; Wagner et al., 1990; Tang et al., 1996; Hara et al., 1997; Ogris et al., 1998). As opposed to their success in vitro, the attempted in vivo use of these agents to delivery DNA has revealed many complications. Notable downsides include those related to toxicity (Wolfert and Seymour, 1996), antigenicity (Stankovics et al., 1994), complement activation (Plank et al., 1996), solubility (Toncheva et al., 1998), blood compatibility (Yang and Huang, 1997), and stability (Kwoh et al., 1999). These complications relate to the size and charge of DNA carrier complexes and ultimately to the molecular characteristics of the carrier itself.
The high molecular weight (HMW) of most DNA carrier polymers increases the likelihood of activating the complement system (Plank et al., 1996), eliciting antigenicity (Stankovics et al., 1994), and being cytotoxic (Wolfert and Seymour, 1996). The size and heterogeneity of such polymers also significantly complicates regio-specific derivatization with ligands or polyethylene glycol (Wolfert et al., 1996) to arrive at optimized well-characterized DNA carriers that mediate efficient gene transfer in vivo.
In an attempt to circumvent the problems of HMW carriers, several low molecular weight (LMW) carrier peptides have been developed. Certain of these even mediate in vitro gene transfer as efficiently as their HMW counterparts (Wadhwa et al., 1997; Plank et al., 1999). These offer the advantage of controlled synthesis and defined purity, which then allows for a strategy of systematic optimization to increase expression levels and eliminate side effects.
However, when tested for in vivo efficacy, LMW carriers have been shown to lack sufficient stability to remain intact during circulation and thereby do not significantly protect DNA from premature metabolism in tissue (Kwoh et al., 1999). Recently, certain crosslinking agents have been applied to form caged DNA condensates by template polymerization, but thus far, these have not been shown to be transfection competent (Trubetskoy et al, 1998; 1999). The use of carriers with different isomeric forms is also being investigated (Laurent et al., 1999). In seeking a solution to the relative instability of LMW carriers, increased stability should not be over-emphasized to the detriment of gene transfer efficiency and/or gene expression.
Therefore, despite increasing attention in this field, the development of effective, low toxicity carriers for DNA delivery still constitutes a major challenge. The generation of low toxicity carriers with sufficient stability to mediate in vivo delivery and yet still provide efficient gene expression in target tissues would be a significant advance.
The present invention overcomes these and other drawbacks inherent in the prior art by providing a range of DNA carrier compositions for use in improved gene transfer methods. The invention particularly provides low molecular weight carriers that are minimal in size, reduce toxicity, function to condense DNA into small particles, have increased stability, mediate gene expression and, preferably, provide surprisingly effective gene expression levels.
The compositions and methods of the invention achieve high affinity binding to DNA using only low molecular weight (LMW) carriers. The invention is thus broadly based upon providing temporary, but sufficient, stability through molecular crosslinking of LMW carriers to DNA condensates.
Certain aspects of the invention exploit unpaired amines to provide effectively crosslinked peptide DNA condensates. Increasing the stability of peptide DNA condensates is thus achieved by introducing dialdehyde groups, such as glutaraldehyde, to crosslink surface amine groups on the peptides. LMW peptides cross-linked in this manner condense DNA into small condensates with improved stability, as demonstrated by increased resistance to shear stress induced fragmentation. Glutaraldehyde-crosslinked condensates are also significantly more resistant to in vitro metabolism by serum endonucleases and still mediate steady-state gene expression.
Important embodiments of the present invention concern LMW peptide DNA condensates with metabolic stability and reversibility, which provide high level gene expression. The LMW peptide portions of the DNA condensates incorporate multiple cysteine residues that allow the peptides to undergo oxidation to form interpeptide disulfide bonds while bound to DNA. Once in a target cell, the disulfide cross-links are reduced, releasing DNA for efficient gene expression. The reducing environment of the endosome is believed to mediate disulfide reduction and DNA release.
In preferred embodiments, the cross-linking peptides of the invention are prepared by replacing lysine residues with cysteine residues to provide low molecular weight DNA condensing peptides that spontaneously cross-link, after binding to DNA, by forming interpeptide disulfide bonds. The peptides thus contain multiple sulfhydryl groups designed to spontaneously polymerize and cross-link when bound to DNA. The stability of cross-linked peptide DNA condensates is dependent, at least in part, on the number of cysteines incorporated into the peptide. Disulfide bond formation in this manner decreases DNA condensates particle size, relative to control peptide DNA condensates, and prevents dissociation of peptide DNA condensates.
Importantly, the gene expression mediated by the cross-linked DNA condensates of the invention is not only maintained, but is actually increased 5 to 60-fold over uncrosslinked DNA condensates, depending on the number of cysteine residues. The cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, indicating a mechanism involving intracellular release of DNA triggered by disulfide bond reduction.
The invention provides panels and an admixtures of low molecular weight, synthetic cross-linking peptides (of generally less than twenty amino acids) that not only form small, stabilized DNA condensates, but mediate efficient gene expression. The xe2x80x9cself cross-linkingxe2x80x9d peptides and resultant DNA condensates of the invention are thus highly efficient DNA delivery agents that represent a breakthrough in gene therapy technology. The peptide-DNA condensates of the invention provide their own multicomponent peptide condensed DNA formulations and can be further combined with other gene therapy agents, such as matrices, carriers and targeting agents, for even more effective in vivo therapies.
Exemplary combined compositions and methods of using the present invention include the formulation of DNA-peptide condensates with matrices that allow cells to migrate into the matrix to encounter and take up the DNA; formulation of DNA-peptide condensates with targeting agents for cellular or sub-cellular delivery; and combination with stealthing agents, such as polyethylene glycol (PEG), to reduce non-specific cellular uptake and/or interaction with blood components, thereby enhancing systemic gene delivery (Ogris et al., 1999).
The cross-linking peptides themselves may be covalently derivatized with polyethylene glycol (PEG). PEG-peptides form a steric layer on the surface of DNA condensates that block optimization, mask DNA condensate recognition by the reticuloendothelial system and increase DNA condensate solubility by blocking the formation of aggregates.
In still further embodiments, the self-cross-linking peptides of the invention may be converted into cross-linking and targeting peptides by the addition of targeting units. For example, target specificity is achieved by derivatizing a cross-linking peptide with a single N-glycan resulting in glycopeptides that direct targeting to either the asialoglycoprotein receptor on hepatocytes or the mannose receptor on liver Kupffer cells.
DNA co-condensates can thus be prepared using systematically determined admix ratios of cross-linking glycopeptide and PEG-peptide. The backbone of cross-linking peptides are chemically modifiable by reduction of the amide linkages to install secondary amines designed to buffer endosomes and allow DNA condensates to release into the cytosol of target cells. Once in the cytosol, cross-linked DNA condensates slowly release plasmid DNA following disulfide reduction. Decreasing DNA metabolism by increasing DNA condensate stability prolongs the liver half-life of DNA and produces a longer duration and higher level of gene expression in vivo. The present invention thus overcomes various limitations of current nonviral gene delivery systems.
The dialdehyde aspects of the present invention provide nucleic acid condensates, comprising a nucleic acid and at least two nucleic acid-binding peptides that are crosslinked via a low molecular weight dialdehyde; nucleic acid condensates, comprising a nucleic acid and at least two nucleic acid-binding peptides that are crosslinked via glutaraldehyde; nucleic acid condensates, comprising a nucleic acid and at least two positively-charged, low molecular weight peptides that are crosslinked via glutaraldehyde; and nucleic acid condensates, comprising a nucleic acid and at least two nucleic acid-binding peptides; wherein the peptides are crosslinked by glutaraldehyde.
Further nucleic acid condensates are those comprising a nucleic acid and at least two low molecular weight peptides with sufficient positive charge to bind to a nucleic acid, the peptides being linked via a glutaraldehyde crosslinker; and comprising a nucleic acid and an amount of glutaraldehyde-crosslinked, nucleic acid-binding peptides that form a non-covalently linked peptide-nucleic acid condensate.
Stable nucleic acid condensates are provided, comprising nucleic acids and an amount of glutaraldehyde-crosslinked, nucleic acid-binding peptides effective to stabilize the nucleic acid. Nucleic acid condensates with in vivo stability comprise a nucleic acid and an amount of glutaraldehyde-crosslinked, nucleic acid-binding peptides effective to stabilize the nucleic acid under in vivo conditions.
Methods of stabilizing a nucleic acid-peptide condensate comprise crosslinking nucleic acid-binding peptides within the condensate with at least a first glutaraldehyde crosslinker; whereas methods of protecting a nucleic acid from degradation comprise preparing a nucleic acid-peptide condensate and crosslinking at least a portion of the peptides within the condensate using a glutaraldehyde crosslinker.
Methods of stabilizing a nucleic acid-peptide condensate comprise crosslinking nucleic acid-binding peptides within the condensate with at least a first glutaraldehyde crosslinker; whereas methods of protecting a nucleic acid from degradation comprise preparing a nucleic acid-peptide condensate and crosslinking at least a portion of the peptides within the condensate using a glutaraldehyde crosslinker.
The self-crosslinking aspects of the invention provide a cationic linker comprising sufficient positive charge to bind to a nucleic acid and at least two thiol groups; a low molecular weight cationic linker comprising sufficient positive charge to bind to a nucleic acid and at least two thiol groups; and cationic linkers wherein the linker comprises a positively-charged peptide, a cationic polymer, or a cationic lipid with sufficient positively-charged amine groups to bind to a nucleic acid.
Nucleic acid condensing agents are provided comprising a low molecular weight cationic linker with sufficient positive charge to bind to a nucleic acid and sufficient thiol groups to form a self-crosslinked construct that induces a bound nucleic acid to condense.
Further provide are peptides comprising sufficient positively-charged residues to bind to a nucleic acid and capable of forming a disulfide-bonded peptide; and peptides comprising sufficient positively-charged residues to bind to a nucleic acid and at least two thiol groups.
The peptides are between about 3 and about 50 amino acids in length; between about 4 and about 50 amino acids in length; between about 5 and about 50 amino acids in length; between about 10 and about 50 amino acids in length; between about 5 and about 40 amino acids in length; between about 5 and about 30 amino acids in length; between about 5 and about 20 amino acids in length; between about 5 and about 10 amino acids in length; between about 25 and about 30 amino acids in length; between about 20 and about 25 amino acids in length; between about 15 and about 20 amino acids in length; and between about 10 and about 15 amino acids in length.
The peptides are further about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or so amino acids in length.
The peptides may comprise between about 2 and about 45 positively-charged residues; between about 3 and about 45 positively-charged residues; between about 4 and about 45 positively-charged residues; between about 5 and about 45 positively-charged residues; between about 10 and about 45 positively-charged residues; between about 15 and about 45 positively-charged residues; between about 20 and about 45 positively-charged residues; between about 25 and about 45 positively-charged residues; between about 30 and about 45 positively-charged residues; between about 35 and about 45 positively-charged residues; between about 40 and about 45 positively-charged residues.
This can be achieved by comprising between about 2 and about 45 positively-charged lysine residues; between about 3 and about 45 positively-charged lysine residues; between about 4 and about 45 positively-charged lysine residues; between about 5 and about 45 positively-charged lysine residues; between about 10 and about 45 positively-charged lysine residues; between about 15 and about 45 positively-charged lysine residues; between about 20 and about 45 positively-charged lysine residues; between about 25 and about 45 positively-charged lysine residues; between about 30 and about 45 positively-charged lysine residues; between about 35 and about 45 positively-charged lysine residues; between about 40 and about 45 positively-charged lysine residues.
The peptides may be thiolylated substantially polylysine peptides. They may comprise at least 3, 4, 5, 6, 7, 8 or so thiol groups or may have only two thiol groups.
At least one, two, three, four, five, six, seven, eight or so cysteine residue may provide at least one of the thiol groups. Two cysteine residues are suitable examples. The peptides may be alkylated, wherein it may be that the at least a first cysteine residue is alkylated. D-amino acid residues may be employed if desired.
The peptides may be dispersed within a pharmaceutically acceptable medium, bound to a nucleic acid, associated with a matrix, associated with a carrier or a targeting ligand, covalently linked to a targeting ligand, covalently linked to at least a first glycosyl unit, thereby forming a glycopeptide targeting ligand, covalently linked to at least a first oligosaccharide unit to form a glycopeptide targeting ligand, or may be both bound to a nucleic acid and associated with a matrix, carrier or a targeting ligand.
The peptides may thus be summarized as being between about 3 and about 50 amino acids in length, comprising sufficient positively-charged residues to bind to a nucleic acid and at least two thiol groups, such as two cysteine residues. The peptides may comprise sufficient positively-charged residues to bind to a nucleic acid and a number of thiol groups sufficient to form a reversibly-linked nucleic acid-peptide composition that dissociates under endosomal conditions.
Nucleic-acid cross-linking peptides may comprise an amount of positively-charged residues effective to bind nucleic acid and at least two thiol groups effective to form spontaneous peptide-crosslinks sufficient to produce ionic-crosslinked nucleic acids upon contact, optionally with at least a first glycosyl unit.
Exemplary nucleic acid condensates are those comprising a nucleic acid and a nucleic acid-binding peptide that comprises a plurality of positively-charged residues and at least two thiol groups and those comprising a nucleic acid condensate that comprises a nucleic acid and a nucleic acid-binding peptide that comprises a plurality of positively-charged residues and at least two thiol groups.
Peptide-linked nucleic acid condensates may comprise nucleic acids and an amount of positively-charged, double-thiol-containing nucleic acid-binding peptides effective to form a non-covalently linked peptide-nucleic acid condensate; or nucleic acids and an amount of positively-charged, double-thiol-containing nucleic acid-binding peptides effective to form interpeptide disulfide bonds, thereby condensing nucleic acids in non-covalent contact with the disulfide-bonded peptides; or a nucleic acid and nucleic acid-binding peptides, wherein the peptides each comprise a plurality of positively-charged residues and at least two thiol groups and form a condensed nucleic acid particle of between about 10 nm and about 20 nm in diameter upon contact with nucleic acids.
Stable nucleic acid condensates are those comprising a nucleic acid and at least two positively-charged nucleic acid-binding peptides that comprise an amount of thiol groups effective to stabilize the nucleic acid; nucleic acid condensates with in vivo stability comprise a nucleic acid and at least two positively-charged nucleic acid-binding peptides that comprise an amount of thiol groups effective to stabilize the nucleic acid under in vivo conditions.
Reversibly-bound nucleic acid-peptide condensates comprise nucleic acids and an amount of positively-charged nucleic acid-binding peptides with an amount of thiol groups effective to form a nucleic acid-peptide condensate that is substantially stable in an extracellular biological environment and that releases nucleic acids upon contact with an intracellular endosome.
Gene delivery complexes of the invention comprise a carrier and a nucleic acid condensate of nucleic acids and positively-charged nucleic acid-binding peptides that comprise an amount of thiol groups effective to condense and stabilize the nucleic acids. The carrier may be a polyethyleneglycol carrier.
Targeted gene delivery complexes comprise a targeting ligand and a nucleic acid condensate of nucleic acids and positively-charged nucleic acid-binding peptides that comprise an amount of thiol groups effective to condense and stabilize the nucleic acids.
Multimolecular complexes of the present invention comprise a carrier, a targeting ligand and a nucleic acid condensate of nucleic acids and positively-charged nucleic acid-binding peptides that comprise an amount of thiol groups effective to condense and stabilize the nucleic acids. The multimolecular complexes may further comprise a biocompatible matrix.
Gene-matrix formulations may comprise a biocompatible matrix and a nucleic acid condensate comprising a nucleic acid and positively-charged nucleic acid-binding peptides that comprise an amount of thiol groups effective to stabilize and condense the nucleic acids.
Stealthed gene-delivery compositions may comprise a stealthing agent and a nucleic acid condensate comprising nucleic acids and positively-charged peptides that bind to nucleic acid and comprise an amount of thiol groups effective to stabilize and condense the nucleic acids.
The unified concepts of the invention thus provide nucleic acid condensates, comprising a nucleic acid and at least a first and second low molecular weight cationic linker bound to the nucleic acid; wherein:
the first and second cationic linker are crosslinked to each other by reaction with a low molecular weight dialdehyde; or
the first and second cationic linker each comprise at least two thiol groups and wherein the cationic linkers are crosslinked to each other by reaction of the thiol groups.
Nucleic acid condensates with a particle size of between about 10 nm and about 100 nm in diameter; between about 10 nm and about 50 nm in diameter; and between about 10 nm and about 20 nm in diameter are included, but are not limiting of the invention.
Preferred cationic linkers are first and second low molecular weight peptides, preferably of between about 6 and about 20 amino acids in length or between about 6 and about 10 amino acids in length or between about 10 and about 20 amino acids in length.
The first and second peptides each preferably comprise between about four and about eight Lysine residues that mediate binding of the peptides to the nucleic acid; and at least two, three or four thiol groups and wherein the peptides are crosslinked to each other by reaction of the thiol groups.
In addition to Cysteine residues, preferred first and second peptides each comprise at least two Penicillamine (Pen) residues that provide the thiol groups. The at least two Cysteine or Penicillamine (Pen) residues are preferably each located at, or proximal to, the termini of the peptides.
At least one of the first or second peptides preferably comprises at least one, two, three, four, five, or six or so secondary or tertiary amine residue that mediates endosomal buffering of the peptide upon uptake into a cell. Histidine residues are suitable examples for endosomal buffering.
Certain preferred peptides have the amino sequence of CWK17C (SEQ ID NO:3), CK4C (SEQ ID NO:9), CK8C (SEQ ID NO:11), CHK6HC (SEQ ID NO:17), PenWK5PenK5PenK5Pen (SEQ ID NO:21) or PenHK4CK4HPen (SEQ IS NO:22).
The purified low molecular weight synthetic peptides themselves form aspects of the present invention, wherein the peptide comprises sufficient positive charge to bind to a nucleic acid and sufficient thiol groups to form disulfide-crosslinked peptides that induce nucleic acids to condense upon contact with a population of the peptides.
A population of purified nucleic-acid condensing peptides is provided, wherein the peptides are synthetic peptides of between about 6 and about 20 amino acids in length, comprise an amount of positively-charged residues effective to bind nucleic acid, comprise at least two thiol groups effective to spontaneously crosslink peptides within the population and comprises an amount of secondary or tertiary amines effective to promote dissociation under endosomal conditions; wherein the population of peptides is effective to form a nucleic acid-peptide condensate that is substantially stable in an extracellular biological environment and that releases nucleic acids intracellularly in a manner effective for gene expression.
Operative associated with at least a first stealthing agent, targeting agent or biocompatible matrix is preferred. The peptides themselves provide for such operative attachment to at least a first stealthing or targeting agent, thereby associating the stealthing or targeting agent with the nucleic acid condensate. Preferred examples are wherein at least one of the first or second peptides comprises a thiol group at each terminus and wherein at least a first stealthing or targeting agent is operatively attached to the peptide at a site distal from each terminus.
Polyethyleneglycol (PEG) stealthing agents, antibody, growth factor and carbohydrate targeting agents are preferred.
Co-condensates are particular preferred, such as those comprising at least a first peptide operatively attached to at least a first stealthing agent and at least a second peptide operatively attached to at least a first targeting agent. Exemplary co-condensates are those comprising a population of peptides; wherein between about 5% and 20% of the peptides are operatively attached to PEG; between about 5% and 20% of the peptides are operatively attached to a glycosyl targeting unit; and the remainder of the peptides comprise about four Histidine or secondary or tertiary amine residues.
The nucleic acids may be single-stranded nucleic acids, double-stranded nucleic acids, degradation-resistant nucleic acids, DNA, plasmid DNA, RNA, and DNA-RNA chimera, an antisense nucleic acid, a ribozyme or an expression vector. Preferably, the nucleic acids express at least a therapeutic product upon provision to a cell.
These include antigenic or immunogenic proteins or polypeptides that stimulate an immune response when expressed by a cell of the immune system; cytotoxic or apoptosis-inducing proteins or polypeptides that induce cell death upon expression in a target cell; a transcription or elongation factor, cell cycle control protein, kinase, phosphatase, DNA repair protein, oncogene, tumor suppressor, angiogenic protein, anti-angiogenic protein, immune response stimulating protein, cell surface receptor, accessory signaling molecule, transport protein, enzyme, anti-bacterial or anti-viral protein or polypeptide.
Further examples are nucleic acids that encode a hormone, neurotransmitter, growth factor, growth factor receptor, interferon, interleukin, chemokine, cytokine, colony stimulating factor or chemotactic factor protein or polypeptide; such as growth hormone (GH), a fibroblast growth factor (FGF), a granulocyte/macrophage colony stimulating factor (GMCSF), an epidermal growth factor (EGF), a platelet derived growth factor (PDGF), an insulin-like growth factor (IGF), a leukemia inhibitory factor (LIF) or an activin/inhibin protein or polypeptide.
At least two distinct nucleic acids, up to and including plurality of nucleic acids may be included.
Kits of the invention comprising, in at least a first container:
a plurality of low molecular weight peptides with sufficient positive charge to bind to a nucleic acid and an amount of glutaraldehyde effective to cross-link at least a portion of the peptides; or
a plurality of low molecular weight peptides that each comprise at least two thiol groups and have sufficient positive charge to bind to nucleic acids, the peptides spontaneously forming intermolecular disulfide-crosslinks.
Methods of preparing a nucleic acid-peptide condensate comprise contacting a nucleic acid with at least two nucleic acid-binding peptides that have sufficient positive charge to bind to a nucleic acid; wherein:
the nucleic acid-binding peptides are cross-linked with glutaraldehyde, thereby condensing the nucleic acid in contact with the crosslinked peptides; or wherein
the nucleic acid-binding peptides each comprise a thiol capacity sufficient to spontaneously form interpeptide crosslinks, thereby condensing the nucleic acid in contact with the crosslinked peptides.
Method of expressing a nucleic acid in a cell comprise contact a cell with an effective amount of a nucleic acid condensate that comprises a nucleic acid having bound thereto at least two low molecular weight nucleic acid-binding peptides; wherein:
the nucleic acid-binding peptides are cross-linked with glutaraldehyde; or wherein
the nucleic acid-binding peptides each comprise at least two thiol groups and spontaneously form disulfide cross-links.
The cell may be located within an animal, wherein the nucleic acid condensate is administered to the animal.
Methods for providing a nucleic acid to an animal comprise providing to the animal an effective amount of a nucleic acid condensate that comprises the nucleic acid in functional association with a population of low molecular weight nucleic acid-binding peptides; wherein:
the nucleic acid-binding peptides are cross-linked with glutaraldehyde in a manner effective to stabilize the nucleic acid under in vivo conditions; or wherein
the nucleic acid-binding peptides each comprise at least two thiol groups and spontaneously form disulfide cross-links in a manner effective to stabilize the nucleic acid under in vivo conditions.
Method for expressing a nucleic acid in target cells of an animal comprise providing to the animal an effective amount of a nucleic acid condensate comprising a nucleic acid that is non-covalently attached to an amount of low molecular weight nucleic acid-binding peptides; wherein:
the nucleic acid-binding peptides are cross-linked with glutaraldehyde in a manner effective to stabilize the nucleic acid under in vivo conditions for a time sufficient to allow delivery of the nucleic acid to the target cells within the animal; or wherein
the nucleic acid-binding peptides each comprise at least two thiol groups and spontaneously form disulfide cross-links in a manner effective to stabilize the nucleic acid under in vivo conditions for a time sufficient to allow delivery of the nucleic acid to the target cells within the animal.
The invention further provides compositions in accordance with the present invention for use in therapy or diagnosis, including in gene therapy and human gene therapy.
Uses of the compositions in accordance with the present invention in the manufacture of medicaments for use in expressing nucleic acids in animals and humans and for use in gene therapy and human gene therapy are further encompassed.